The present invention relates to a microarray slide for detecting, identifying and quantifying toxic algae. More specifically, the microarray, which comprises DNA barcodes for each toxic alga, is configured to detect and quantify an assemblage of toxic algae from marine environmental samples to a high confidence level to meet EU Directive 2002/225/EC for the quantification of toxic algae in marine coastal waters as a means of determining fishery closure. It provides an alternative to the mouse bioassay for fishery closure, which has been banned by the EU for ethical reasons since 2012. The need for invoking the more expensive HPLC method for toxin determination can be reduced with a reliable molecular method that can identify and quantify toxic algae.
The world's oceans cover 70 percent of the Earth's surface, and their dominant populations, both numerically and biomass-wise, belong to microscopic protests (including microalgae) and prokaryotes. Microalgae in marine and brackish waters of Europe regularly cause harmful effects, considered from the human perspective, in that they cause economic damage to fisheries and tourism and health issues. These episodes encompass a broad range of phenomena collectively referred to as <((harmful algal blooms)) (HABs) or red tides. For adequate management of these phenomena, monitoring of microalgae is essential and is required by EU directive 2002/225/EC for all European countries with a marine coastline.
The global scale of toxin producing micro-algae should not be underestimated. For example, the most serious would be the numbers of human intoxications with ciguatera, caused by the dinoflagellate Gambierdiscus, is currently estimated at some 50,000 per year. Every year, 1-2 human deaths are linked to the ingestion of PSP toxins caused by Alexandrium. Although these problems are restricted to the tropical/warm temperate sphere of the globe, it demonstrates the urgent need to be able to monitor and prevent toxic HAB events. With global warming warm water species are now moving into north temperate European waters. In Europe, this is affected through a series of directives that require coastal member states to monitor water for toxin producing species and their toxins in shellfish. Starting with the EU Shellfish Hygiene Directive 91/492/EEC, a series of Directives was issued to include newly discovered toxins, and stipulating the methods of analysis and maximum permitted levels in shellfish. The most important of these are 2002/225/EC and 2074/2005 (pertaining to toxin levels and analysis and methods) and more recently 15/2011 (analysis methods).
The natural occurrence of toxin producing algae, and the continual human demand for shellfish consumption, means that the need for their monitoring is here to stay.
The cost of this monitoring of plankton and toxins is enormous. Although there is limited ‘hard’ information on the economic impact of HABs, a relatively recent study in the US (Anderson et al., 2000) has estimated, on a national basis, that:                the cost of monitoring is equivalent to 5% annual shellfish industry turnover        the cost of lost harvest and damaged product caused by contamination with biotoxins is 5% of industry turnover        the public health costs caused by lost working days, hospitalisations etc. add another 5% of annual turnover        
In Europe, similar information is also difficult to uncover, but the context is well set if one takes the case of Ireland where the shellfish aquaculture production currently runs at €47 million annually (Bowne et al., 2007) and the budget for the Irish National Biotoxin and Toxic Phytoplankton monitoring programme, carried out under the auspices of the Food Safety Authority of Ireland, and operated through the Irish Marine Institute, is €1.7 million, representing ˜3.5% of annual industry turnover. Similarly, Scottish shellfish production is valued at ˜£20 million, the most part of which is through culture of the edible mussel Mytilus edulis, and the monitoring programmes, run by the Food Standards Agency Scotland, has a budget of just under £2 million.
Clearly the development of an industry that is both natural and sustainable, but which has such a heavy financial burden, requires all possible assistance in order to overcome such ‘natural hazards’ as toxic HABs, because the (natural) problems caused by toxicity will never go away. Approximately 2000 water samples are analysed annually in Ireland as part of the National Monitoring Programme (NMP). This requires a staff of 4 people, augmented slightly during the busy summer months. Most samples are scanned for toxic/harmful species but samples from 10 sites (out of a total of ˜60) are analysed for their total phytoplankton community. Light microscopy is the routine analysis method, each sample requiring ca. 2 hours on average to examine. Comparable figures for other monitoring programmes are annual throughputs of 1000 samples (Scotland), 5000 samples (REPHY, France), and 6000 samples (Galicia, Spain). These figures reflect a work rate of processing some 20 samples per week per person. The number of man-hours involved in the monitoring process is clearly enormous. Often the results are available up to 5 days after taking the sample making mitigation strategies almost impossible. This invention seeks, inter alia, to provide a solution to this problem.
Present day monitoring is time consuming and based on morphology as determined by light microscopy is insufficient to give definitive species and toxin attribution. Molecular techniques, which are faster and more reliable, would reduce the number of inevitable mistakes caused by human error that is an ever-present facet of this type of work. Of particular relevance are the situations with respect to Pseudo-nitzschia, which cannot be identified to species level using light microscopy, and Alexandrium, another genus with which it is also virtually impossible to identify accurately to species using this technique. Identification and quantification to a level of accuracy is essential if toxic blooms are to be accurately forecast to allow their mitigation and fishery closure enforced only when needed to avoid unnecessary economic loss and because toxic and non-toxic strains of the same species, i.e., Alexandrium, overlap in their distribution.
The advent of molecular biological techniques has greatly enhanced our ability to analyse all organisms. These techniques are slowing making inroads into monitoring for toxic algae in terms of monitoring for the presence of a species and the toxins they produce. One approach that is extensively used in such studies is to identify species by specific molecular probes or barcodes. In hybridisation experiments, these probes can therefore be used to identify species of interest by binding to the target's sequence and later detection by a probe-attached label. Calibration curves based on culture material can be generated to convert the probe signal intensity from its label to cell numbers, thus meeting EU requirements for toxic algal monitoring using cell numbers as the trigger level for fisheries closure or before initiating tests for toxins. The microarray presented here can be universally applied to monitor for toxic algae in any country with toxic algal blooms. In Japanese waters, the toxic algae causing the most problems will not the same as those along the western and eastern coasts of Australia and North America, or along the western coasts of Europe, thus it is advantageous to have universal barcodes that specifically detect all variations of each toxic algal species.